ABSTRACT
High glucose (HG) is one of the basic factors of diabetic nephropathy (DN), which leads to high morbidity and disability. During DN, the expression of glomerular glucose transporter 1 (GLUT1) increases, but the relationship between HG and GLUT1 is unclear. Glomerular mesangial cells (GMCs) have multiple roles in HG-induced DN. Here, we report prominent glomerular dysfunction, especially GMC abnormalities, in DN mice, which is closely related to GLUT1 alteration. In vivo studies have shown that BBR can alleviate pathological changes and abnormal renal function indicators of DN mice. In vitro, BBR (30, 60 and 90 µmol/L) not only increased the proportion of G1 phase cells but also reduced the proportion of S phase cells under HG conditions at different times. BBR (60 µmol/L) significantly reduced the expression of PI3K-p85, p-Akt, p-AS160, membrane-bound GLUT1 and cyclin D1, but had almost no effect on total protein. Furthermore, BBR significantly declined the glucose uptake and retarded cyclin D1-mediated GMC cell cycle arrest in the G1 phase. This study demonstrated that BBR can inhibit the development of DN, which may be due to BBR inhibiting the PI3K/Akt/AS160/GLUT1 signalling pathway to regulate HG-induced abnormal GMC proliferation and the cell cycle, supporting BBR as a potential therapeutic drug for DN.
Subject(s)
Berberine , Diabetes Mellitus , Diabetic Nephropathies , Animals , Berberine/pharmacology , Cell Cycle , Cell Division , Cell Proliferation , Diabetes Mellitus/pathology , Diabetic Nephropathies/pathology , Glucose/metabolism , Glucose Transporter Type 1/genetics , Glucose Transporter Type 1/metabolism , Mesangial Cells/metabolism , Mice , Phosphatidylinositol 3-Kinases/metabolism , Proto-Oncogene Proteins c-akt/metabolismABSTRACT
AIMS: This study aimed to investigate the characteristics and mechanism of autophagy on podocyte apoptosis under high glucose (HG) conditions and further explore the effect of berberine on podocyte autophagy, apoptosis and the potential mechanism. MATERIALS AND METHODS: The levels of LC3II/I in podocytes stimulated with HG were detected at 0, 2, 4, 8, 12, 24, 36 and 48 h by western blotting. CCK-8 was used to detect the viability of podocytes. The level of autophagy was detected by western blotting, transmission electron microscopy and immunofluorescence. Podocyte apoptosis was analysed by using Hoechst staining, western blotting, annexin V/propidium iodide dual staining, and confocal microscopy. Then, podocytes were transfected with siRNA to silence mTOR, and the expression levels of proteins and mRNA involved in the mTOR/P70S6K/4EBP1 pathway were further investigated by western blotting and qRT-PCR. KEY FINDINGS: In this study, we found significantly reduced LC3II/LC3I and increased p62 in podocytes stimulated with HG for 24 h, and the level of autophagy reached a minimum at 24 h. Berberine restored podocyte viability and significantly attenuated HG-mediated inhibition of autophagy, as evidenced by the increased expression of LC3II/LC3I, the number of autophagosomes and the inhibition of p62. Moreover, berberine counteracted HG-induced podocyte apoptosis and injury, which was negatively correlated with the autophagy effect. Notably, silencing mTOR with siRNA augmented the inhibition of P70S6k and 4EBP1 phosphorylation, which was similar to the effect of berberine. SIGNIFICANCE: Berberine activates podocyte autophagy by inhibiting the mTOR/P70S6K/4EBP1 signaling pathway, thereby alleviating podocyte apoptosis.